Unbiased and accurate cloning is essential for analysis of the human genome and other genomes important to our well-being. Complete genomic sequencing and comparative genomics reveal new information about metabolic pathways in normal tissue as well as diseased states, such as cancer, diabetes, aging, AIDS, and others. Genomic sequencing also provides critical information about horizontal gene transfer, evolution of protein families, and the genetic repertoire and evolution of species. However, current methods of constructing libraries and cloning individual genes are often extremely challenging, time consuming, and costly. Importantly, they also leave many uncloned gaps. These obstacles obscure important information about specific genes and genomic regions (e.g., centromeres) and limit the scope of genomes studied. The objectives of this proposal are to provide new vectors and streamlined methods to create clone libraries that surpass current standards of fidelity, complexity, lack of cloning bias, and ease of analysis. These techniques will provide access to existing clone gaps in the human genome and allow cloning of other recalcitrant genes and genomes. Specific aims include development of a novel linear cloning vector and demonstration of its capacity for stable maintenance of otherwise "unclonable" sequences, such as inverted repeats, microsatellite repeats, and large clones of genomic DNA (>8 kb) from AT-rich pathogens, such as Pneumocystis carinii. The aims also include developing new methods of PCR amplification and rapid production of sequencing template from single bacterial colonies. These methods will be optimized for bench-scale and high-throughput amplification and sequencing. The advances proposed by this research will increase the accuracy and lower the costs of cloning and sequencing single genes or entire genomic or cDNA libraries. These advances will allow researchers to readily analyze DNA regions that are currently difficult or impossible to study. Improved sequence information will accelerate the advances in biotechnology and increase the potential of individual and comparative genomic analyses. [unreadable] [unreadable] [unreadable]